Interactions of Tetrahymena dynein with microtubule protein. Tubulin-induced stimulation of dynein ATPase activity

The ATPase (EC 3.6.1.3) activity of 30 S dynein from Tetrahymena cilia was remarkably stimulated by porcine brain tubulin at pH 10. The activity increased with increasing concentration of tubulin until the molar ratio of tubulin dimer to 30 S dynein reached approx. 10. The optimum of the ATPase activity of 30 S dynein in the presence of tubulin was 1?2 mM for MgCl₂ and 2 mM for CaCl₂. Increasing ionic strength gradually inhibited the stimulation effects of tubulin. Activation energies of 30 S dynein in the presence and absence of tubulin were almost the same. At the temperatures beyond 25 °C stimulation effects of tubulin disappeared. ATP was a specific substrate even in the presence of tubulin. In kinetic investigations parallel reciprocal plots were observed in a constant ratio of divalent cations to ATP of 2, indicating that tubulin was less tightly bound to 30 S dynein in the presence of ATP than in the absence. The similar results were obtained at pH 8.2. 14 S dynein and the 12 S fragment which have poor ability to recombine with outer fibers were also activated with brain tubulin.     

Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication.

The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules

Dynein was obtained by high salt extraction of Tetrahymena cilia and purified by DEAE-Sephacel chromatography. This fraction consisted of a mixture of 30 S dynein (80%) and the 14 S ATPase (15%). The column purification effectively removed tubulin and adenylate kinase. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the 30 S dynein was composed of a major heavy chain (approximately 400 kD, three copies), three intermediate chains (70, 85, and 100 kD), and a group of light chains (approximately 20 kD). The binding of the column-purified dynein to bovine brain microtubules was characterized as follows. (i) Titration of the dynein with microtubules showed a linear increase in turbidity up to an equivalence point of 2.7 mg of dynein/mg of tubulin with apparently tight binding; (ii) the addition of ATP caused the turbidity of the solution of decrease to a level equal to the sum of free dynein plus microtubules; (iii) transmission electron microscopy indicated that microtubules were decorated with dynein arms spaced at a 24-nm longitudinal repeat and that the dynein decoration was removed upon addition of ATP; (iv) cross-section images of microtubules that were saturated with dynein showed six to seven dynein arms around a microtubule consisting of 14 protofilaments, corresponding to a molar ratio of one dynein/six tubulin dimers; (v) the dynein arms were bound primarily by their broader end which corresponds to the end normally bound to the B-subfiber in vivo. Experiments with purified 30 and 14 S dyneins indicated that the dynein-microtubule binding activity and the ATP-induced dissociation were the properties of the 30 S dynein alone. These studies demonstrate that the 30 S dynein under our conditions (50 mM PIPES, pH 6.96, 4 mM MgSO4) interacts with bovine brain microtubules through the ATP-sensitive site of the dynein arm.     

Bull sperm 19S dynein polymerizes brain tubulin into microtubules

Crude dynein extracted from bull sperm flagella polymerized pure phosphocellulose tubulin isolated from brain tissues into microtubules. This effect was predominantly due to the 19S dynein particle in the extract. ATP stimulated up to five fold the polymerization of brain tubulin by bull sperm dynein. Hydrolysis of ATP was not required since vanadate at a concentration sufficient to block dynein ATPase activity did not interfere with ATP stimulation and because the non hydrolyzable ATP analogue adenylyl (beta-gamma-methylene) diphosphate (AMPPCP) had effects similar to those of ATP. These results suggest that, in addition to hydrolyzing ATP to generate the driving force necessary for microtubule sliding within the axoneme, dynein may also interact with ATP to polymerize tubulin into microtubules.     

Effects of adenosine triphosphate on N-ethylmaleimide-induced modification of 30S dynein from Tetrahymena cilia.

Ciliary 30S dynein of Tetrahymena was investigated with regard to modification of the ATPase activity with N-ethylmaleimide (NEM) in the presence of ATP. The elevation of enzyme activity due to the modification was largely repressed by addition of ATP at a concentration of 1 mM or more during preincubation of 20 h at 0 degrees C. The repression was highly specific for ATP, though ADP and AMPPNP showed slight repressive effects. After complete hydrolysis of ATP added to the preincubation mixture, however, elevation of 30S dynein ATPase activity occurred. It is suggested that the repression by ATP of NEM-induced elevation of 30S dynein ATPase activity is simply due to a protecting effect of ATP on certain SH group(s) (probably SH1-type group(s)) around the active center of 30S dynein. When 30S dynein was maximally activated by modification with NEM, ATP or ADP did not significantly promote the inactivation of the modified enzyme upon further treatment with NEM, indicating that 30S dynein lacks the characteristics of SH2-type groups. On the other hand, ATP also showed a protective effect against inhibition of native 30S dynein by high concentrations of NEM. High concentrations of ADP and AMPPNP were inhibitory to 30S dynein ATPase activity but inorganic phosphate did not inhibit 14S or 30S dynein ATPase activities at all.     

Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.     

A high-affinity ATP-binding site on 30S dynein

The enhancing effect of low concentrations (eg, 8 μM) of bis(4-fluoro-3-nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1–3 μM ATP. Adenylylimidodiphosphate (AMP-PNP) is less effective than ATP, while β,γ-methylene-adenosine triphosphate (AMP-PCP), though a much stronger inhibitor of ATPase activity than AMP-PNP, does not protect against enhancement. These results demonstrate the presence of a high-affinity ATP-binding site on 30S dynein.     

Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C)

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.     

The alkaline adenosine triphosphatase activity of 30S dynein after modification of the SH groups. Possible involvement of some of the most reactive SH groups.

An apparent 'triphasic' alteration of 30S dynein ATPase activity was produced by treatment with various amounts of NEM when the modification and susequent ATPase assay were carried out at pH 7.4 and pH 10-10.2, respectively. The Mg-ATPase activity was markedly inhibited by modification of the most reactive SH groups with 10 microM NEM, although the same treatment had no significant effect on the activity when assayed at neutral pH. Increasing the NEM concentration to 0.3 mM largely restored the enzyme activity, but a further increase in NEM concentration inhibited the enzyme activity again. This unusual response of 30S dynein ATPase at pH 10-10.2 was accounted for by the results of Arrhenius plots of the enzyme activity at pH 10.1; the enzyme protein modified with not more than 10 microM NEM was not stable under the assay conditions (pH 10-10.2 at 25 degrees C), whereas modification with 0.3 mM NEM stabilized 30S dynein against the assay conditions. The possible significance of the 10 microM NEM-induced inhibition of the 30S dynein alkaline ATPase activity is discussed in connection with the participation of SH groups of 30S dynein in the enzyme activity.     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.     

Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases

The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Activation of the dynein adenosinetriphosphatase by cross-linking to microtubules

The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.     

The substrate specificity of dynein from Tetrahymena cilia

The substrate specificity of the 22S dynein ATPase from Tetrahymena cilia was investigated. The 22S dynein exhibited a high specificity for ATP in terms of both apparent Km and Vmax: naturally occurring nucleoside triphosphates other than ATP were hydrolyzed slowly with an apparent Km of 0.25-1 mM, a sharp contrast to that of ATP hydrolysis (1-4 microM). Pyrophosphate was a poor inhibitor for the dynein ATPase, indicating weak affinity. Since dynein binds ATP tightly and hydrolyzes it at a high rate, a method to determine a trace amount of ATP in the presence of other nucleoside triphosphates has been developed by taking advantage of this enzymatic characteristic of dynein. The effect of P1,P5-di(adenosine-5'-)-pentaphosphate (Ap5A) on the 22S dynein ATPase was also investigated. Ap5A acted as a weak competitive inhibitor of the ciliary 22S dynein ATPase and the nonlinearity of the double-reciprocal plot of the ATPase was confirmed in the presence of Ap5A.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Vinblastin

Vinblastine, a plant alkaloid which inhibits tubulin polymerization, stimulated an ATPase activity in microtubules. When microtubule proteins were separated into microtubule-associated proteins (MAPs) and tubulin by phosphocellulose column chromatography, vinblastine did not stimulate an ATPase activity recovered in the MAPs fraction unless tubulin was present. Therefore, vinblastine is considered to act through its binding to the tubulin molecule on MAPs ATPase. Divalent cations that activate tubulin-dependent MAPs ATPase activity were also required for the stimulation by vinblastine. In the presence of Ca2+ and vinblastine the ATPase activity was most active and the extent of stimulation reached about 200% of the original level in the absence of vinblastine. Half-maximal stimulation was attained when the molar ratio of vinblastine to tubulin was 0.5. The concentration of tubulin for half-maximal stimulation was increased in the presence of vinblastine, while divalent cation requirements were decreased. Several factors such as KCl (100 mM), alkaline pH (pH 7.5), and low temperature (10 degrees C) were not responsible for the disappearance of the stimulation. Vincristine stimulated tubulin-dependent MAPs ATPases activity as vinblastine did, whereas the activity was scarcely affected by colchicine, podophyllotoxin, strychnine, and chlorpromazine. Actin had no effect on MAPs ATPase activity in the absence and presence of vinblastine when it was used in place of tubulin.     

Interactions of dynein arms with b subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphosphate

Tetrahymena 30S dynein was extracted with 0.5 M KCl and tested for retention of several functional properties associated wtih its in situ force-generating capacity. The dynein fraction will rebind to extracted outer doublets in the presence of Mg2+ to restore dynein arms. The arms attach at one end to the A subfiber and form bridges at the other end to the B subfiber of an adjacent doublet. Recombined arms retain an ATPase activity that remains coupled to potential generation of interdoublet sliding forces. To examine important aspects of the dynein- tubulin interaction that we presume are directly related to the dynein force-generating cross-bridge cycle, a simple and quantitative spectrophotometric assay was devised for monitoring the associations between isolated 30S dynein and the B subfiber. Utilizing this assay, the binding of dynein to B subfibers was found to be dependent upon divalent cations, saturating at 3 mM Mg2+. Micromolar concentrations of MgATP2- cause the release of dynein from the B subfiber; however, not all of the dynein bound under these conditions is released by ATP. ATP- insensitive dynein binding results from dynein interactions with non-B- tubule sites on outer-doublet and central-pair microtubules and from ATP-insensitive binding to sites on the B subfiber. Vanadate over a wide concentration range (10(-6)-10(-3) M) has no effect on the Mg2+- induced binding of dynein or its release by MgATP2-, and was used to inhibit secondary doublet disintegration in the suspensions. In the presence of 10 microM vanadate, dynein is maximally dissociated by MgATP2- concentrations greater than or equal to 1 microM with half- maximal release at 0.2 microM. These binding properties of isolated dynein arms closely resemble the cross-bridging behavior of in situ dynein arms reported previously, suggesting that quantitative studies such as those presented here may yield reliable information concerning the mechanism of force generation in dynein-microtubule motile systems. The results also suggest that vanadate may interact with an enzyme- product complex that has a low affinity for tubulin.     

Tryptic fragmentation of 30-S dynein from Tetrahymena cilia.

30-S dynein ATPase from Tetrahymena cilia was digested with trypsin (dynein: trypsin = 20:1, by weight) at 25 degrees C for 20 min, resulting in the release of a 12-S fragment possessing ATPase activity. The 12-S ATPase fraction obtained by sucrose gradient centrifugation contained several polypeptide chains as indicated by SDS gel electrophoresis. The largest chain was smaller than the subunit of 30-S dynein and almost the same size as 14-S dynein. On the other hand, when 14-S dynein was digested in a similar manner, its sedimentation value changed from 14 to 12 S, but the peak of ATPase activity was retained at 14 S, suggesting differences in amino acid sequences between the 30 and 14-S dyneins. When the time course of tryptic digestion of 30-S dynein was investigated in a trypsin:dynein ratio of 1:200, discrete fragmentation took place, producing an intermediate fragment of 24 S and the 12-S fragment. The 24-S fragment recombined with outer fibers to some extent, while the 12-S fragment lacked this ability. However, the 12-S fragment was somewhat stimulated to recombine with outer fibers in the presence of other components involved in the trypsin digest. The enzymatic characteristics of the 12-S fraction were different from those of 30-S dynein, especially the activity dependence on pH showing a typical bell-shaped curve.